Physiological role of hepatic M3 mAChRs studied with mutant mouse models Many studies support the concept that an increase in the rate of hepatic glucose production is the major determinant of fasting hyperglycemia in type 2 diabetes. A better understanding of the signaling pathways and molecules that regulate hepatic glucose metabolism is therefore of great clinical importance. Several studies suggest that an increase in vagal outflow to the liver leads to decreased hepatic glucose production and reduced blood glucose levels. ACh is the major neurotransmitter of the vagus nerve and exerts its parasympathetic actions via activation of mAChRs. We therefore decided to study the potential physiological roles of hepatocyte mAChRs in regulating glucose homeostasis. RT-PCR studies showed that the M3 mAChR is the only mAChR subtype expressed by mouse liver/hepatocytes. To assess the physiological role of hepatocyte M3 mAChRs in vivo, we used gene targeting (Cre/loxP) and transgenic techniques to generate mutant mice lacking or overexpressing M3 receptors in hepatocytes only. Detailed in vivo phenotyping studies failed to reveal any significant metabolic differences between the M3 receptor mutant mice and their control littermates, independent of whether the mice were fed regular chow or a high-fat diet. Moreover, the expression levels of genes for various key transcription factors, signaling molecules, and enzymes regulating hepatic glucose fluxes were not significantly altered in the M3 receptor mutant mice. These data suggest that the pronounced metabolic effects that can be observed following activation of afferent hepatic vagal nerves are mediated by non-cholinergic signaling pathways. Since the activity of efferent hepatic vagal nerves is predicted to be critically involved in maintaining normal blood glucose levels, the identification of other (non-muscarinic) hepatic signalling pathways that are under vagal control may lead to novel strategies to modulate hepatic glucose fluxes for therapeutic purposes. A novel chemical-genetic strategy to study G protein regulation of beta cell function in vivo Type 2 diabetes has emerged as one of the major threats to human health worldwide. Impaired function of pancreatic beta cells is one of the key hallmarks of type 2 diabetes, and therapies targeted at improving beta cell function are predicted to offer considerable therapeutic benefit. Beta cell function is modulated by the actions of different classes of heterotrimeric G proteins, and beta cells are known to express many different GPCRs. The functional consequences of activating specific beta cell G protein signaling pathways in vivo are not well understood at present, primarily due to the fact that beta cell GPCRs are also expressed by many other tissues. Thus, the availability of an experimental system in which specific G protein families can be activated in a beta cell-specific and temporally controlled fashion would be highly desirable. To address this issue, we developed a novel chemical-genetic strategy that allows for the conditional and selective activation of distinct beta cell G proteins in intact animals. Specifically, we created two lines of transgenic mice each of which expressed a specific designer GPCR (at similar expression levels) in beta cells only. Importantly, the two M3 mAChR-based receptors differed in their G protein-coupling properties (Gq/11 versus Gs). The two designer receptors could be efficiently activated by clozapine-N-oxide (CNO), an otherwise pharmacologically inert compound, but not by ACh, the endogenous M3 mAChR ligand. As a result, CNO treatment of the newly developed transgenic mice led to the activation of beta cell Gq/11 or Gs signaling pathways in vivo, in a conditional and &#946;cell-selective fashion. We found that conditional and selective activation of beta cell Gq/11 signaling in vivo leads to striking increases in both first- and second-phase insulin release, greatly improved glucose tolerance in obese, insulin-resistant mice, and elevated beta cell mass, associated with pathway-specific alterations in islet gene expression levels. Selective stimulation of beta cell Gs triggered qualitatively similar in vivo metabolic effects. Thus, this newly developed chemical-genetic strategy represents a powerful tool to study G protein regulation of beta cell function in vivo. In principal, this new technology can be applied to study the physiological and pathophysiological relevance of distinct G protein signaling pathways in virtually every cell type. RGS4 and spinophilin as negative regulators of M3 mAChR-mediated insulin release The M3 mAChR is a prototypic class I Gq-coupled receptor. Previous work from our lab supports the concept that strategies aimed at enhancing signaling through M3 mAChRs expressed by pancreatic beta cells may be beneficial for the treatment of type 2 diabetes by promoting glucose-stimulated insulin secretion (GSIS;Gautam et al., Cell Metab. 3, 449-461, 2006). To identify factors that regulate M3 receptor activity in insulin-producing cells, we used the murine insulinoma cell line, MIN6, as a model system. Treatment of MIN6 cells with the muscarinic agonist, oxotremorine-M (Oxo-M), led to a pronounced increase in intracellular calcium levels and GSIS. We demonstrated, by using siRNA technology, that these responses were primarily mediated by M3 mAChRs. qRT-PCR studies showed that RGS4 (RGS = Regulator of G protein Signaling) was highly expressed in MIN6 cells and mouse pancreatic islets. Interestingly, siRNA-mediated knockdown of RGS4 expression greatly enhanced Oxo-M-mediated increases in intracellular calcium levels and GSIS in MIN6 cells. In contrast, siRNA-mediated knockdown of RGS4 expression had smaller effects on insulin responses mediated by activation of other Gq-coupled receptors, including distinct P2Y and vasopressin receptor subtypes. Finally, siRNA-mediated knockdown of spinophilin also enhanced Oxo-M-induced responses in MIN6 cells, in a fashion similar to RGS4 knockdown. Spinophilin is a scaffolding protein known to link the M3 mAChR to members of the R4 subfamily of RGS proteins. In summary, RGS4 or spinophilin knockdown leads to enhanced M3 receptor-mediated increases in GSIS in a mouse insulinoma cell line. These findings may lead to new strategies for the therapy of type 2 diabetes aimed at enhancing signaling through beta cell M3 mAChRs.